Background: Enhancer of zeste homolog 2 (EZH2) is a component of Polycomb Repressive Complex 2 (PRC2) and a key epigenetic regulator. EZH2 is overexpressed in metastatic prostate cancer (PCa), and its activity is critical for PCa progression and therapeutic resistance. Inhibition of EZH2 has become a promising cancer treatment option. Studies showed that agents depleting EZH2 protein had better antitumor efficacy than those only inhibiting its methyltransferase activity, suggesting that combination therapy may lead to more effective by inhibiting both EZH2 activity and protein turnover. SRC tyrosine kinase is upregulated in castration resistant prostate cancer (CRPC), and plays a driver role in PCa progression. Despite a promising improvement in Phase II clinical trials, combination therapy using docetaxel and the SRC inhibitor dasatinib failed in a later large randomized phase III trial- suggesting that patient selection and new drug combination are critical for development of effective treatment. Hypothesis and Aims: Our preliminary study showed that SRC can induce EZH2 tyrosine phosphorylation. The phosphorylation mutant compromised EZH2 stability and activity. The mutant failed to interact with PRC2 associated proteins and de-repressed EZH2-regulated genes. Furthermore, combination treatment of EZH2 and SRC inhibitors displayed synergistic growth inhibitory effects. We hypothesize that tyrosine phosphorylation of EZH2 is critical for its oncogenic activity and contributes to castration resistance; and simultaneous inhibition of EZH2 and its upstream kinases may attenuate CRPC growth more effectively. Specific Aims: 1) Delineate the mechanisms by which EZH2 activity is regulated by tyrosine phosphorylation. 2) Determine the functional significance of EZH2 phosphorylation in resistance to androgen deprivation therapy in preclinical models. Study Design: We will study how EZH2 tyrosine phosphorylation modulates its stability and functional interaction with its known partners, and identify phosphorylation-dependent interacting partners using proteomic approaches. We will also perform ChIP-seq to define the signature of genes regulated by phosphorylated EZH2 using the anti-pEZH2 phospho-specific antibody we developed. We will examine whether EZH2 phosphorylation mutation or tyrosine kinase inhibitors can sensitize CRPC to EZH2 inhibitors in xenograft models. Successful completion of this project will provide new mechanistic insights into castration resistance and lead to the development of new effective combination therapy to circumvent therapeutic resistance in prostate cancer treatment, and optimally reduce or delay lethality in Veterans who suffer from prostate cancer.